Biophysics Seminar 3 TT5

Talk 1: Docking site-mediated photostabilization for single-molecule and super-resolution imaging
Cindy Close, Tinnefeld Group, LMU Munich

DNA-PAINT is a single-molecule localization microscopy technique, relying on transient hybridization of fluorescently labeled single-stranded DNA imager strands to complementary docking strands on target molecules.1 During acquisition, docking sites are imaged over the course of multiple binding, dissociation and photobleaching events. Through constant imager strand exchange, the limited photon budget of a single fluorophore is circumvented, making it possible to extract super-resolution images at high laser illumination intensities. Over long periods of continuous high-duty cycle excitation of fluorophores, DNA-PAINT binding sites can, however, be depleted.2 Fluorophores in triplet excited states may generate singlet oxygen and downstream reactive oxygen species (ROS), damaging the docking sites and labeled target structures (Figure 1a). The use of triplet state quenchers (TSQ) and enzymatic scavenging systems is further limited to systems insensitive to pH change or high additive concentration. Inspired by fluorophore regeneration and self-repair mechanisms, we link the TSQ cyclooctatetraene to a DNA sequence.3,4 This photostabilizer strand binds directly next to the imager at the docking site, thereby allowing for self-regeneration and programmed exchange (Figure 1b). The presented contribution shows how this approach can increase the accessible photon budget. The method is characterized in a DNA origami model structure and applied to image microtubules in fixed cells. The improved longevity of DNA-PAINT docking sites is shown and the impact of photostabilizer strand regeneration is explored. The ability to mix and match optimal photostabilizer/dye pairs in this modular approach could be beneficial e.g., for multi-color measurements, that often require multiple rounds of imaging.

Talk 2: Breaking the Concentration Barrier in Single-Molecule Fluorescence with Fluorogenic Probes – a Universal Approach
Mirjam Kümmerlin, Kapanidis Group, Biophysics & Kavli Institute for Nanoscience Discovery (Oxford)

The “high concentration barrier” of ~50 nM fluorescent species is one of the main limitations of single-molecule fluorescence measurements – overcoming it will advance many in vitro and in vivo single-molecule applications, including tracking in crowded environments, super-resolution imaging, and smFRET experiments. One way to generate good data from high-concentration environments is by employing fluorogenic probes (i.e. labels that become fluorescent upon binding to a target).
We have implemented strategies to achieve fluorogenicity in ssDNAs, which we use to label targets carrying a complementary strand. The quenching efficiency and fluorescence enhancement upon duplex formation can be tailored through the use of several fluorophore-quencher combinations, label lengths, and buffer compositions. These allow for single-molecule experiments at concentrations of up to 10 µM fluorescent labels – a 200-fold improvement without the need for any special optics or nanofabrication. Our detailed understanding of the quenching processes allows us to develop fluorogenic labels for a multitude of experimental applications, without being limited by length, sequence of the label, or spectral regions of the fluorescence. To demonstrate the plug-and-play level solution this can offer, we demonstrate DNA-PAINT super-resolution imaging of viral particles using a fluorogenic 6nt long, fluorogenic imager. To further highlight new experimental paths only possible with fluorogenic labels, we perform smFRET measurements over extraordinarily long observations spans of up to one hour for a single molecule, circumventing photo-bleaching through constant exchange of fluorogenic ssDNAs supplying donor and acceptor dyes (REFRESH-FRET).